Brad C. Bennett

Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins.

Significance Membrane proteins (MPs) perform a variety of essential cellular functions, account for about one-third of encoded proteins in genomes, and comprise more than one-half of human drug targets. High-resolution structures are essential to understand the underlying molecular mechanisms of MPs and facilitate structure-based drug design efforts. Detergents are indispensible in the solubilization of MPs, but they tend to destabilize MPs and often impede the growth of well-ordered protein crystals. We describe a class of structurally unique detergents, designated as facial amphiphiles, which improved MP stability and success in the crystallization of different families of MPs. Amphiphile selection is a critical step for structural studies of membrane proteins (MPs). We have developed a family of steroid-based facial amphiphiles (FAs) that are structurally distinct from conventional detergents and previously developed FAs. The unique FAs stabilize MPs and form relatively small protein–detergent complexes (PDCs), a property considered favorable for MP crystallization. We attempted to crystallize several MPs belonging to different protein families, including the human gap junction channel protein connexin 26, the ATP binding cassette transporter MsbA, the seven-transmembrane G protein-coupled receptor-like bacteriorhodopsin, and cytochrome P450s (peripheral MPs). Using FAs alone or mixed with other detergents or lipids, we obtained 3D crystals of the above proteins suitable for X-ray crystallographic analysis. The fact that FAs enhance MP crystallizability compared with traditional detergents can be attributed to several properties, including increased protein stability, formation of small PDCs, decreased PDC surface flexibility, and potential to mediate crystal lattice contacts.

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate.

Hydrogen atoms play a central role in many biochemical processes yet are difficult to visualize by x-ray crystallography. Spallation neutron sources provide a new arena for protein crystallography with TOF measurements enhancing data collection efficiency and allowing hydrogen atoms to be located in smaller crystals of larger biological macromolecules. Here we report a 2.2-Å resolution neutron structure of Escherichia coli dihydrofolate reductase (DHFR) in complex with methotrexate (MTX). Neutron data were collected on a 0.3-mm3 D2O-soaked crystal at the Los Alamos Neutron Scattering Center. This study provides an example of using spallation neutrons to study protein dynamics, to identify protonation states directly from nuclear density maps, and to analyze solvent structure. Our structure reveals that the occluded loop conformation [monomer (mon.) A] of the DHFR·MTX complex undergoes greater H/D exchange compared with the closed-loop conformer (mon. B), partly because the Met-20 and β(F-G) loops readily exchange in mon. A. The eight-stranded β sheet of both DHFR molecules resists H/D exchange more than the helices and loops. However, the C-terminal strand, βH, in mon. A is almost fully exchanged. Several D2Os form hydrogen bonds with exchanged amides. At the active site, the N1 atom of MTX is protonated and thus charged when bound to DHFR. Several D2Os are observed at hydrophobic surfaces, including two pockets near the MTX-binding site. A previously unidentified D2O hydrogen bonds with the catalytic D27 in mon. B, stabilizing its negative charge.

An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca2+ blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca2+. The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.

Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography.

Significance There is immense difficulty in mapping out the complete details of an enzyme’s mechanism, especially those that catalyze an acid-base reaction, owing to the simple fact that hydrogen atom positions are rarely known with any confidence. Ultrahigh-resolution X-ray and, better still, neutron crystallography can provide this crucial layer of information. We paired these techniques to reveal the catalytic mechanism of dihydrofolate reductase (DHFR), an enzyme necessary for nucleotide biosynthesis and a classical drug target. In a complex that closely resembles the catalytically active state, DHFR stabilizes a particular substrate conformer and likely elevates the pKa of the substrate atom that is protonated. This protonation occurs directly via water, with its access to the substrate regulated by structural fluctuation of the enzyme. Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP+. The neutron data were collected to 2.0-Å resolution using a 3.6-mm3 crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.

A quantized mechanism for activation of pannexin channels

Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels.

Protein structures by spallation neutron crystallography

The capabilities of the Protein Crystallography Station at Los Alamos Neutron Science Center for determining protein structures by spallation neutron crystallography are illustrated, and the methodological and technological advances that are emerging from the Macromolecular Neutron Crystallography consortium are described.

On the determinants of amide backbone exchange in proteins: a neutron crystallographic comparative study.

The hydrogen/deuterium-exchange (HDX) method, coupled with neutron diffraction, is a powerful probe for investigating molecular dynamics. In the present report, general determinants of HDX are proposed based on 12 deposited neutron protein structures. The parameters that correlate best with HDX are the depth within the protein structure of the amide N atom and the secondary-structure type. Both the B factor of the amide N atom and the ratio B/B correlate moderately. However, solvent accessibility only correlates strongly for one molecule and hydrogen-bonding distance correlates for two molecules with respect to amide HDX. In addition to the relatively small number of neutron structures available, the limitations to this type of analysis, namely resolution, data completeness and the data-to-parameter ratio, are discussed briefly. A global analysis of HDX was performed to overcome some of these obstacles, damping the effects of outliers and the extreme variation of the data sets arising from resolution limitations. From this, amide depth and hydrogen-bonding distance to the amide (a measure of interaction strength) show strong global correlation with HDX. For some structures, the constituents of the hydrophobic protein core could be identified based on contiguous regions that are resistant to exchange and have significant depth. These may, in fact, constitute minimal folding domains.

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.

Background Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C2-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis. Methodology/Principal Findings Using His-HDX-MS, the pK a values and the half-lives (t 1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK a and t 1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK a, t 1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK a and t 1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings. Conclusions/Significance Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

Function and dynamics of macromolecular complexes explored by integrative structural and computational biology

Three vignettes exemplify the potential of combining EM and X-ray crystallographic data with molecular dynamics (MD) simulation to explore the architecture, dynamics and functional properties of multicomponent, macromolecular complexes. The first two describe how EM and X-ray crystallography were used to solve structures of the ribosome and the Arp2/3-actin complex, which enabled MD simulations that elucidated functional dynamics. The third describes how EM, X-ray crystallography, and microsecond MD simulations of a GPCR:G protein complex were used to explore transmembrane signaling by the β-adrenergic receptor. Recent technical advancements in EM, X-ray crystallography and computational simulation create unprecedented synergies for integrative structural biology to reveal new insights into heretofore intractable biological systems.

Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP+.

A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ of 4×1.3×0.7 mm (3.6 mm3) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 Å resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 Å resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR-folate-NADP+ ternary-complex structure in order to examine the protonation state, protein dynamics and solvent structure of the complex, furthering understanding of the catalytic mechanism.